Magnetic microspheres for use in fluorescence-based applications

ABSTRACT

Microspheres, populations of microspheres, and methods for forming microspheres are provided. One microsphere configured to exhibit fluorescent and magnetic properties includes a core microsphere and a magnetic material coupled to a surface of the core microsphere. About 50% or less of the surface of the core microsphere is covered by the magnetic material. The microsphere also includes a polymer layer surrounding the magnetic material and the core microsphere. One population of microspheres configured to exhibit fluorescent and magnetic properties includes two or more subsets of microspheres. The two or more subsets of microspheres are configured to exhibit different fluorescent and/or magnetic properties. Individual microspheres in the two or more subsets are configured as described above.

PRIORITY APPLICATION

This application claims priority to U.S. Ser. No. 60/645,549 entitled“Magnetic Microspheres for use in Fluorescence-Based Application” filedJan. 20, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to magnetic microspheres for usein fluorescence-based applications. Certain embodiments relate to amicrosphere that includes a magnetic material coupled to a surface of acore microsphere and a polymer layer surrounding the magnetic materialand the core microsphere.

2. Description of the Related Art

The following description and examples are not admitted to be prior artby virtue of their inclusion in this section.

Magnetic microspheres are currently used in a wide variety ofapplications, including: hyperthermic treatment of tumors; directeddelivery of therapeutic substances to target locations in livingsystems; cell, polynucleotide, and protein isolation; and clinicalanalysis of biomolecules. Microspheres suitable for such purposes areavailable from a number of commercial sources in a number of differentconfigurations. These microspheres often include a magneticallysusceptible substance and a spherical matrix material such as an organicpolymer or silica. The microspheres may have several configurations suchas a magnetic core surrounded by a matrix; small magnetic particlesdispersed throughout a matrix; and a magnetic coating on the outside ofa spherical matrix. Each of these microsphere configurations hasadvantages and disadvantages, and selection of the appropriateconfiguration is dependent on the intended use of the microspheres.

For many purposes, appropriate microspheres display paramagnetism orsuperparamagnetism, rather than ferromagnetism. Such microspheres havenegligible magnetism in the absence of a magnetic field, but applicationof a magnetic field induces alignment of the magnetic domains in themicrospheres, resulting in attraction of the microspheres to the fieldsource. When the field is removed, the magnetic domains return to arandom orientation so there is no interparticle magnetic attraction orrepulsion. In the case of superparamagnetism, this return to randomorientation of the domains is nearly instantaneous, while paramagneticmaterials will retain domain alignment for some period of time afterremoval of the magnetic field. This retention of domain alignment maylead to microsphere aggregation in the absence of an external magneticfield, which is often undesirable. Ferromagnetic materials havepermanently aligned domains, so microspheres including such magneticmaterials will readily aggregate.

The matrix material associated with the magnetic material also variesdepending on the intended use of the microspheres, with silica andpolymer latex being the most commonly used matrix materials. Bothmaterials can be used to create substantially uniform magneticmicrospheres in a wide range of diameters. Magnetic silica microspheresare often more stable over a wider range of temperatures thanmicrospheres made from organic polymers such as polystyrene, and bothmaterials may decompose in certain environments such as acidic oraromatic solvents. Additionally, silica microspheres are often moredense than latex microspheres, which can be an important considerationin the choice of magnetic microsphere matrices.

A significant and growing use of magnetic microspheres is in the fieldof biological assays. Assays for proteins and oligonucleotides can beperformed on the surface of the microspheres, which can then bemagnetically separated from the reaction mixture before thecharacteristics of the microspheres are measured. Isolation of the assaymicrospheres prior to measurement decreases interference of non-targetmolecules with the measurements thereby producing more accurate results.

Concurrent with the increasing interest in magnetic microspheres forbiological assays is the development of assays conducted on fluorescentmicrospheres. The use of fluorescent labels or fluorescent materialcoupled to a surface of the microspheres or incorporated into themicrospheres allows preparation of numerous sets of microspheres thatare distinguishable based on different dye emission spectra and/orsignal intensity. In a biological assay, the fluorescence and lightscattering of these microspheres can be measured by a flow cytometer oran imaging system, and the measurement results can be used to determinethe size and fluorescence of the microspheres as well as thefluorescence associated with the assay system being studied (e.g., afluorescently labeled antibody in a “capture sandwich” assay), asdescribed in U.S. Pat. No. 5,948,627 to Lee et al., which isincorporated by reference as if fully set forth herein. By varying theconcentrations of multiple dyes incorporated in the microspheres,hundreds, or even thousands, of distinguishable microsphere sets can beproduced. In an assay, each microsphere set can be associated with adifferent target thereby allowing numerous tests to be conducted for asingle sample in a single container as described in U.S. Pat. No.5,981,180 to Chandler et al., which is incorporated by reference as iffully set forth herein.

Fluorescently distinguishable microspheres may be improved by renderingthese microspheres magnetically responsive. Examples of methods forforming fluorescent magnetic microspheres are described in U.S. Pat. No.5,283,079 to Wang et al., which is incorporated by reference as if fullyset forth herein. The methods described by Wang et al. include coating afluorescent core microsphere with magnetite and additional polymer ormixing a core microsphere with magnetite, dye, and polymerizablemonomers and initiating polymerization to produce a coated microsphere.These methods are relatively simple approaches to the synthesis offluorescent magnetic microspheres, but are not suitable for creating thelarge numbers of precisely dyed microspheres used in relatively largemultiplex assays such as those as described in U.S. Pat. No. 5,981,180to Chandler et al.

This limitation of the methods of Wang et al. is due to the fact thatmost fluorescent dye molecules are extremely sensitive to attack byradical species generated during radical initiation polymerizations. Ifthese radicals inactivate even a relatively small number of dyemolecules, precise quantities of dye in the microspheres cannot beachieved. Furthermore, if the methods of Wang et al. are used tosynthesize non-fluorescent magnetic microspheres, and dyeing of themicrospheres is attempted using the solvent swelling method described inU.S. Pat. No. 6,514,295 to Chandler et al., which is incorporated byreference as if fully set forth herein, a relatively large amount of themagnetic material will be released from the microspheres during dyeingsince the magnetic material is not chemically bound to the microspheres.In particular, physical entrapment of the magnetic material in themicrospheres will be disrupted by the swelling process, and the magneticmaterial will be released into solution.

Fluorescent magnetic microspheres are also described in U.S. Pat. No.6,268,222 to Chandler et al., which is incorporated by reference as iffully set forth herein. In this method, nanospheres are coupled to apolymeric core microsphere, and the fluorescent and magnetic materialsare associated with either the core microsphere or the nanospheres. Thismethod produces microspheres with desirable characteristics, but thenanosphere-microsphere bond may be susceptible to cleavage under severereaction conditions. A coating surrounding the microsphere andnanospheres bound thereto may be used to improve this association but,again, the use of radical initiators to form this coating can compromisethe fluorescent emission profile of the microsphere.

A more desirable configuration for a fluorescent magnetic microsphere isa magnetically responsive microsphere that can be dyed using establishedtechniques, such as those described in U.S. Pat. No. 6,514,295 toChandler et al. In general, this method uses solvents that swell themicrosphere thereby allowing migration of the fluorescent material intothe microsphere. These dyeing solvents include one or more organicsolvents. Therefore, the microspheres must be able to tolerate organicsolvents without losing their compositional integrity. Additionally,free magnetite interferes with a number of biological reactions.Therefore, microspheres that are susceptible to loss of even arelatively small amount of magnetite are unacceptable. As such, magneticmicrospheres should be constructed such that the magnetic material istightly bound to the micro spheres thereby preventing loss of themagnetic material during the swelling process.

Magnetic microspheres are described in U.S. Pat. No. 5,091,206 to Wanget al., U.S. Pat. No. 5,648,124 to Sutor, and U.S. Pat. No. 6,013,531 toWang et al., which are incorporated by reference as if fully set forthherein. However, the microspheres produced by the methods disclosed inthese patents to Wang et al. may lose magnetic material when disposed inorganic solvents since the magnetic material is not chemicallyimmobilized. In each of these patents, substantially small particles ofmagnetic material are coated on a polymeric core microsphere, with apolymeric shell as an outer coating. The magnetic particles aresynthesized and processed to minimize particle size. Some magneticmaterials, such as Fe₃O₄, progress from ferromagnetic to paramagnetic tosuperparamagnetic as particle size decreases. Therefore, magnetiteparticles having the smallest size possible are used to form themicrospheres such that the product microsphere shows little magneticretentivity.

However, if the product microsphere is also configured to emit afluorescent signal from dye substances in the core microsphere, thethickness of the layer of magnetic particles on the surface of themicrosphere core becomes an important consideration. For example, sincemost magnetic substances are optically opaque, a relatively thickcoating of magnetic particles on the surface of the microsphere corewill cause excessive light scatter or blocking of photon transmission.Using the methods of these patents, in which the magnetic component isdesigned to have the minimum particle size, magnetic content would haveto be limited to allow light transmission through the magneticcomponent. In fact, to provide a magnetic microsphere having a diameterof 7 μm and 5% magnetic content, the entire surface of the coremicrosphere would have to be coated with a layer of magnetite having athickness of 15 nm. This thickness results in a significantly lowerfluorescent signal. Even if some of the magnetic particles are larger,as described by Sutor, the presence of a relatively large number ofsmaller magnetic particles will strongly impact the emission profile ofthe microsphere. One object of the invention of Sutor is to providemagnetically responsive microparticles having more electro magneticunits (EMUs) per gram of material than known microparticles.

The previously used methods also use surfactants and stabilizers in thepreparation of the outer coatings. For many purposes, the presence ofthese molecules on the surface of the microsphere is acceptable.However, when used in bioassays, the surfactants can result in unwantedinterference and changes in the binding efficiency of biomolecules tothe microsphere surface. Washing procedures may reduce the quantity ofsurfactants and stabilizers associated with the microsphere surface, butcompletely removing the surfactants and stabilizers is extremelydifficult.

Therefore, it would be a significant improvement over existingtechnologies to provide methods for forming a microsphere containinggreater than about 2% by weight of a magnetically responsive material,without significantly hindering light transmission into and out of themicrosphere. It would be a further improvement if this magneticallyresponsive material is strongly associated with the microsphere therebyreducing loss of the magnetically responsive material during dyeing andis surrounded by a polymer to substantially prevent the magneticallyresponsive material from interacting with biomolecules of interest.Furthermore, previously used methods can be improved if the outermostpolymer layer is formed in the absence of surfactants and stabilizers.

SUMMARY OF THE INVENTION

The following description of various embodiments of microspheres,populations of microspheres, and methods for forming microspheres is notto be construed in any way as limiting the subject matter of theappended claims.

An embodiment relates to a microsphere configured to exhibit fluorescentand magnetic properties. The microsphere includes a core microsphere anda magnetic material coupled to a surface of the core microsphere. About50% or less of the surface of the core microsphere is covered by themagnetic material. In addition, the microsphere includes a polymer layersurrounding the magnetic material and the core microsphere.

In one embodiment, the core microsphere includes one or more functionalgroups coupled to the surface of the core microsphere. In anotherembodiment, the microsphere includes one or more fluorochromes. In adifferent embodiment, the microsphere includes two or more differentfluorochromes. In some embodiments, the fluorochrome(s) included in themicrosphere are incorporated into the polymer layer as well as the coremicrosphere.

In some embodiments, the magnetic material includes particles having asize of about 10 nm to about 1000 nm. In a preferred embodiment, themagnetic material includes particles having a size of about 50 nm toabout 300 nm. In some embodiments, the magnetic material includes singlecrystals of magnetite. In a further embodiment, the magnetic materialincludes aggregates of particles. For instance, the magnetic materialmay be aggregates of particles smaller than those described above suchthat the aggregates have a size in one of the above ranges. In anotherembodiment, the magnetic material includes a mixed metal magneticmaterial. In an additional embodiment, the microsphere includes one ormore functional groups coupled to an outer surface of the polymer layer.

In one embodiment, the microsphere includes an additional magneticmaterial coupled to an outer surface of the polymer layer and anadditional polymer layer surrounding the additional magnetic material.It will be easily recognized that alternating layers of magneticmaterial and polymer may be repeated in the microsphere until thedesired magnetic content for the microsphere is achieved. In thismanner, the microsphere may include one or more magneticmaterial/polymer layers, each of which is configured such that less thanabout 50% of the surface of the core microsphere is covered withmagnetic material.

In one such embodiment, the magnetic material and the additionalmagnetic material have substantially the same composition. In adifferent embodiment, the magnetic material and the additional magneticmaterial have different compositions. In a further embodiment, at leastone of the magnetic material and the additional magnetic materialincludes a mixed metal magnetic material. In another such embodiment,the polymer layer and the additional polymer layer are formed ofsubstantially the same polymerizable material. In other embodiments, thepolymer layer and the additional polymer layer are formed of differentpolymerizable materials. In some embodiments, the microsphere includesone or more functional groups coupled to an outer surface of theadditional polymer layer. Each of the embodiments of the microspheredescribed above may be further configured as described herein and formedaccording to method embodiments described herein.

The microsphere embodiments described above provide several advantagesover currently used microspheres having magnetic and fluorescentproperties. For example, the microsphere described above can includegreater than about 2% by weight of the magnetic material withoutsignificantly hindering light transmission into and out of themicrosphere. In particular, it has been determined that when more thanabout 50% of the surface of the core microsphere is obscured by themagnetic material, the fluorescent emission of the microsphere becomessignificantly impacted (i.e., the fluorescent emission is significantlylower). In addition, the magnetic material can be strongly associatedwith the microsphere as described further herein such that the magneticmaterial is not released from the microsphere during dyeing. Themagnetized core microsphere is also coated with a polymer layer, whichsubstantially prevents interaction between the magnetic material andbiomolecules of interest. Furthermore, the polymer layer may be formedin the absence of surfactants and stabilizers.

Another embodiment relates to a population of microspheres configured toexhibit fluorescent and magnetic properties. The population includes twoor more subsets of microspheres configured to exhibit differentfluorescent properties, different magnetic properties, or differentfluorescent and magnetic properties. Individual microspheres in the twoor more subsets include a core microsphere and a magnetic materialcoupled to a surface of the core microsphere. About 50% or less of thesurface of the core microsphere is covered by the magnetic material. Theindividual microspheres also include a polymer layer surrounding themagnetic material and the core microsphere. The individual microspheresand the population described above may be further configured asdescribed herein.

An additional embodiment relates to a method for forming microspheresthat exhibit magnetic properties. The method includes combining coremicrospheres with a magnetic material such that the magnetic materialcouples to a surface of the core microspheres to form magnetized coremicrospheres. About 50% or less of the surface of the core microspheresis covered by the magnetic material. The method also includes combiningthe magnetized core microspheres with one or more polymerizablematerials such that the one or more polymerizable materials form apolymer layer surrounding the magnetized core microspheres therebyforming the microspheres that exhibit magnetic properties.

In one embodiment, the method includes separating magnetic particles bysize into a first group and a second group. A substantial portion of themagnetic particles in the first group have a size of about 10 nm orgreater. A substantial portion of the magnetic particles in the secondgroup have a size of about 10 nm or lower. In one such embodiment, themagnetic material combined with the core microspheres includes the firstgroup of magnetic particles. In another embodiment, the method includescombining the formed microspheres with an additional magnetic materialsuch that the additional magnetic material couples to an outer surfaceof the polymer layer and forming an additional polymer layer surroundingthe additional magnetic material.

In some embodiments, the method includes swelling the formedmicrospheres in a fluorochrome containing solvent such that thefluorochrome migrates into the formed microspheres. Such a method alsoincludes changing one or more properties of the fluorochrome containingsolvent such that the formed microspheres shrink thereby entrapping thefluorochrome in the formed microspheres. In a different embodiment, themethod includes incorporating one or more fluorochromes into the coremicrospheres prior to combining the core microspheres with the magneticmaterial. In another embodiment, the method includes coupling one ormore functional groups to an outer surface of the polymer layer. Each ofthe embodiments of the method described above may include any otherstep(s) described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages of the present invention may become apparent to thoseskilled in the art with the benefit of the following detaileddescription of the preferred embodiments and upon reference to theaccompanying drawings in which:

FIG. 1 is a schematic diagram illustrating one example of a system thatmay be used to perform measurements, experiments, and assays with themicrosphere and population embodiments described herein;

FIG. 2 is a schematic diagram illustrating a cross-sectional view of oneembodiment of a magnetized core microsphere that includes a magneticmaterial coupled to a surface of a core microsphere;

FIG. 3 is a schematic diagram illustrating a cross-sectional view of oneembodiment of a microsphere that includes the magnetized coremicrosphere of FIG. 2 surrounded by a polymer layer;

FIG. 4 is a schematic diagram illustrating a cross-sectional view of oneembodiment of the microsphere of FIG. 3, which includes an additionalmagnetic material coupled to an outer surface of the polymer layer; and

FIG. 5 is a schematic diagram illustrating a cross-sectional view of oneembodiment of the microsphere of FIG. 4, which includes an additionalpolymer layer surrounding the additional magnetic material.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and may herein be described in detail. Thedrawings may not be to scale. It should be understood, however, that thedrawings and detailed description thereto are not intended to limit theinvention to the particular form disclosed, but on the contrary, theintention is to cover all modifications, equivalents and alternativesfalling within the spirit and scope of the present invention as definedby the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Microsphere embodiments described herein may serve as vehicles formolecular reactions. Examples of such molecular reactions andexperiments, measurements, and assays in which the microsphereembodiments described herein may be used are described in U.S. Pat. No.5,736,330 to Fulton, U.S. Pat. No. 5,981,180 to Chandler et al., U.S.Pat. No. 6,449,562 to Chandler et al., U.S. Pat. No. 6,524,793 toChandler et al., U.S. Pat. No. 6,592,822 to Chandler, and U.S. Pat. No.6,939,720 to Chandler et al., which are incorporated by reference as iffully set forth herein. The term “microsphere” as used herein isgenerally defined as a composite structure that may or may not bespherical in shape. The terms “microsphere,” “particle,” and “bead” arecommonly used interchangeably by those of ordinary skill in the art.Therefore, the term “microsphere” as used herein may be replaced by theterm “particle” or “bead” without altering the scope of the embodimentsdescribed herein.

FIG. 1 illustrates one example of a system that may be used to performmeasurements, experiments, and assays with the microsphere andpopulation embodiments described herein. It is noted that the figuresdescribed herein are not drawn to scale. In particular, the scale ofsome of the elements of the figures is greatly exaggerated to emphasizecharacteristics of the elements. In addition, the same referencenumerals are used in the figures to indicate elements that may besimilarly configured.

In FIG. 1, the system is shown along a plane through the cross-sectionof cuvette 12 through which microspheres 10 flow. The cuvette may be aquartz or fused silica cuvette such as that used in flow cytometers. Anyother suitable type of viewing or delivery chamber, however, may also beused to deliver the sample for measurement. Microspheres 10 may beconfigured according to embodiments described herein.

The system includes light source 14. Light source 14 may include anyappropriate light source known in the art such as a laser. The lightsource may be configured to emit light having one or more wavelengthssuch as blue light or green light. Light source 14 is configured toilluminate the microspheres as they flow through the cuvette. Theillumination may cause the microspheres to emit fluorescent light havingone or more wavelengths or wavelength bands. In some embodiments, thesystem may include one or more lenses (not shown) configured to focuslight from the light source onto the microspheres or the flowpath. Thesystem may also include more than one light source. In one embodiment,the light sources may be configured to illuminate the microspheres withlight having different wavelengths or wavelength bands (e.g., blue lightand green light). In some embodiments, the light sources may beconfigured to illuminate the microspheres at different directions.

Light scattered forwardly from the microspheres is directed to detectionsystem 16 by folding mirror 18 or another suitable light directingcomponent. Alternatively, detection system 16 may be placed directly inthe path of the forwardly scattered light. In this manner, the foldingmirror or other light directing components may not be included in thesystem. In one embodiment, the forwardly scattered light is lightscattered by the microspheres at an angle of about 180° from thedirection of illumination by light source 14, as shown in FIG. 1. Theangle of the forwardly scattered light may not be exactly 180° from thedirection of illumination such that incident light from the light sourcemay not impinge upon the photosensitive surface of the detection system.For example, the forwardly scattered light may be light scattered by themicrospheres at angles less than or greater than 180° from the directionof illumination (e.g., light scattered at an angle of about 170°, about175°, about 185°, or about 190°).

Light scattered by the microspheres at an angle of about 90° from thedirection of illumination may also be collected. In one embodiment, thisscattered light is separated into more than one beam of light by one ormore beamsplitters or dichroic mirrors. For example, light scattered atan angle of about 90° to the direction of illumination may be separatedinto two different beams of light by beamsplitter 20. The two differentbeams of light may be separated again by beamsplitters 22 and 24 toproduce four different beams of light. Each of the beams of light may bedirected to a different detection system, which may include one or moredetectors. For example, one of the four beams of light may be directedto detection system 26. Detection system 26 may be configured to detectlight scattered by the micro spheres.

Scattered light detected by detection system 16 and/or detection system26 may generally be proportional to the volume of the microspheres thatare illuminated by the light source. Therefore, output signals ofdetection system 16 and/or detection system 26 may be used to determinea diameter or size of the microspheres. In addition, the output signalsof detection system 16 and/or detection system 26 may be used toidentify more than one microsphere that are stuck together or that arepassing through the illumination zone at approximately the same time.Therefore, such microspheres may be distinguished from other samplemicrospheres and calibration microspheres.

The other three beams of light may be directed to detection systems 28,30, and 32. Detection systems 28, 30, and 32 may be configured to detectfluorescence emitted by the microspheres. Each of the detection systemsmay be configured to detect fluorescence of a different wavelength or adifferent range of wavelengths. For example, one of the detectionsystems may be configured to detect green fluorescence. Another of thedetection systems may be configured to detect yellow-orangefluorescence, and the other detection system may be configured to detectred fluorescence.

In some embodiments, spectral filters 34, 36, and 38 are coupled todetection systems 28, 30, and 32, respectively. The spectral filters maybe configured to block fluorescence of wavelengths other than that whichthe detection systems are configured to detect. In addition, one or morelenses (not shown) may be optically coupled to each of the detectionsystems. The lenses may be configured to focus the scattered light oremitted fluorescence onto a photosensitive surface of the detectors.

The detector's output current is proportional to the fluorescent lightimpinging on it and results in a current pulse. The current pulse may beconverted to a voltage pulse, low pass filtered, and then digitized byan analog-to-digital (A/D) converter. Processor 40 such as a digitalsignal processor (DSP) integrates the area under the pulse to provide anumber that represents the magnitude of the fluorescence. As shown inFIG. 1, processor 40 may be coupled to detection system 26 viatransmission medium 42. Transmission medium 42 may include anyappropriate transmission medium known in the art. Processor 40 may alsobe coupled to detection system 26 indirectly via transmission medium 42and one or more other components (not shown) such as an A/D converter.The processor may be coupled to other detection systems of the system ina similar manner.

In some embodiments, the output signals that are responsive tofluorescence emitted by the micro spheres are used to determine anidentity of the micro spheres and information about a reaction taking ortaken place on the surface of the microspheres. For example, outputsignals of two of the detection systems may be used to determine anidentity of the microspheres, and output signals of the other detectionsystem may be used to determine a reaction taking or taken place on thesurface of the microspheres. Therefore, the selection of the detectionsystems and the spectral filters may vary depending on the type of dyesincorporated into or bound to the microspheres and/or the reaction beingmeasured (i.e., the dye(s) incorporated into or bound to the reactantsinvolved in the reaction).

The detection systems that are used to determine an identity of thesample microspheres (e.g., detection systems 28 and 30) may be avalanchephotodiodes (APDs), photomultiplier tubes (PMTs), or otherphotodetectors. The detection system that is used to identify a reactiontaking or taken place on the surface of the microspheres (e.g.,detection system 32) may be a PMT, an APD, or another form ofphotodetector. The system may be further configured as described herein.

Although the system of FIG. 1 is shown to include two detection systemshaving two different detection windows for distinguishing betweenmicrospheres having different dye characteristics, it is to beunderstood that the system may include more than two such detectionwindows (i.e., 3 detection windows, 4 detection windows, etc.). In suchembodiments, the system may include additional beamsplitters andadditional detection systems having other detection windows. Inaddition, spectral filters and/or lenses may be coupled to each of theadditional detection systems.

In another embodiment, the system includes two or more detection systemsconfigured to distinguish between different materials that are reactedon the surface of the microspheres. The different reactant materials mayhave dye characteristics that are different than the dye characteristicsof the microspheres.

The system shown in FIG. 1 may also include a number of other components(not shown) that can be used to measure one or more magnetic propertiesof the microspheres. For example, the system may be configured to excitethe microspheres magnetically as well as optically. The microspheres maybe excited magnetically by applying an external magnetic field to themicrospheres. The external magnetic field may be applied to themicrospheres using any suitable device known in the art. The system mayalso include any appropriate magnetic detector known in the art that canbe used to detect the magnetic field of the microspheres. The magneticfield caused by the induced magnetization of a microsphere that isdetected by the magnetic detector is proportional to the magnetizationof the microsphere, which is in turn proportional to the appliedmagnetic field. The detected magnetic fields of the microspheres can beused to determine an identity of the microspheres and/or a reactiontaking or taken place on the surface of the microspheres.

Microspheres that exhibit measurable magnetic properties provideadvantages over non-magnetic microspheres. For instance, the magneticproperties of the microspheres may be substantially stable over timeparticularly when the magnetic material is protected from exposure toother materials such as reagents (e.g., by a polymer layer as describedherein) and since magnetic labels are not subject to photo-bleaching bythe measurement systems. Furthermore, a substantial magnetic backgroundis not usually present in a sample being analyzed using themicrospheres. In this manner, the sample will not contribute to noise inthe measurements of the magnetic properties of the microspheres. Inaddition, magnetic field application and detection are not blocked byaqueous reagents or biomaterials. Additionally, magnetism may be used toremotely manipulate the microspheres (e.g., for isolation of particularanalytes in a sample). Moreover, a number of relatively sensitivemagnetic field detection devices suitable for biotechnology applicationsare commercially available and can be incorporated into any of thesystems described herein.

Additional examples of systems that may be used to perform measurements,experiments, and assays with microsphere and population embodimentsdescribed herein are illustrated in U.S. Pat. No. 5,981,180 to Chandleret al., U.S. Pat. No. 6,046,807 to Chandler, U.S. Pat. No. 6,139,800 toChandler, U.S. Pat. No. 6,366,354 to Chandler, U.S. Pat. No. 6,411,904to Chandler, U.S. Pat. No. 6,449,562 to Chandler et al., and U.S. Pat.No. 6,524,793 to Chandler et al., which are incorporated by reference asif fully set forth herein. The system described herein may be furtherconfigured as described in these patents. In addition, systems that canbe used to perform measurements of microsphere and populationembodiments described herein include systems described and illustratedin U.S. Patent Application Ser. No. 60/719,010 by Roth filed Sep. 21,2005 and Ser. No. 11/305,805 by Phillips filed Dec. 16, 2005, which areincorporated by reference as if fully set forth herein. The systemsdescribed in these patents and patent applications may includecomponents such as those described above such that the systems canmeasure one or more magnetic properties of the microspheres.

According to one embodiment, a method for forming microspheres thatexhibit magnetic properties includes combining core microspheres with amagnetic material such that the magnetic material couples to a surfaceof the core microspheres to form magnetized (or “magneticallyresponsive”) core microspheres. Core microspheres suitable for use inthe embodiments described herein are available from a number ofcommercial sources and may be formed of any material that will bind tothe selected magnetic material. The bond between the core microspheresand the magnetic material may be covalent, ionic, electrostatic, or anyother suitable bond type known in the art. Some appropriate materialsthat can be used for core microspheres include, but are not limited to,polymers of styrene, divinyl benzene, silica, or acrylamide. The size ofthe core microspheres (e.g., a diameter in the case of spherical coremicrospheres) may be selected based on the measurement system and/ormethod in which the microspheres will be used. Appropriate sizes forcore microspheres range from about 1 μm to about 100 μm in diameter, butother sizes will work as well. It is also noted that greater uniformityin core microsphere size leads to more uniformity in dye (e.g.,fluorescent dye or fluorophore) uptake and magnetic material binding.

In one embodiment, the core microspheres include one or more functionalgroups coupled to the surface of the core microspheres. Surfacefunctionalities can be selected from a number of different reactivemoieties such as amines, thiols, carboxylic acids, hydrazine, halides,alcohols, aldehydes, and any combination thereof. This list offunctionalities is not meant to be a complete list of functionalities,and the microsphere functionalities may include any others known in theart. The one or more functional groups may be coupled to the surface ofthe core microspheres during formation of the core microspheres (e.g.,by polymerization with one or more polymerizable materials that includeone or more functional groups). Alternatively, the one or morefunctional groups may be attached to the surface of the coremicrospheres after the core microspheres have been formed. Suchattachment of the one or more functional groups may be performed usingany suitable method known in the art.

In one embodiment, combining core microspheres with a magnetic materialas described above includes treating a solution of core microsphereswith a solution containing a magnetically responsive material. In oneembodiment, the method includes separating magnetic particles by sizeinto a first group and a second group. A substantial portion of themagnetic particles in the first group have a size of about 10 nm orgreater. A substantial portion of the magnetic particles in the secondgroup have a size of about 10 nm or lower. In one such embodiment, themagnetic material combined with the core microspheres includes the firstgroup of magnetic particles. In such an embodiment, the magneticmaterial combined with the core microspheres does not include the secondgroup of magnetic particles. In this manner, the magnetically responsivematerial may be treated prior to treatment of the core microspheres toeliminate relatively fine particles from the solution that is used totreat the core microspheres.

Magnetic particle size selection can be performed, for example, by apartial acid digestion of the magnetic material (although this step isoptional) followed by filtration or centrifugation to separate largerparticles from smaller particles. The larger particles are retained forcoupling to core microspheres, and the finer particles are discarded. Ithas been shown that repeated washing of the magnetic particles isadvantageous since repeated washing more efficiently removes the finerparticles from the bulk magnetite solution. Other methods that can beused to remove only the finer particles (e.g., particles smaller thanabout 10 nm or particles smaller than about 20 nm) can also be used toprovide magnetic particles suitable for the embodiments describedherein. The washed magnetic particles may then be re-suspended,preferably in an alcoholic solvent, prior to combination with the coremicrospheres. Alcoholic solvents are preferred for re-suspension sincethe reactivity of aqueous solvents and magnetic materials may generatehydroxides or other reduced species on the surface of the magneticmaterial, some of which are known to fluoresce, which would beundesirable for many applications in which the microsphere andpopulation embodiments described herein may be used.

Some advantages of the embodiments described herein are provided by theprocessing of the magnetic material after its synthesis and prior tocoupling to the core microspheres. Prior art methods such as thosedescribed in U.S. Pat. No. 5,283,079 to Wang et al. and U.S. Pat. No.5,648,124 to Sutor, which are incorporated by reference as if fully setforth herein, use techniques to minimize particle size and isolate thefinest particles. For example, the raw magnetite is often partiallydigested with an acid, and the magnetite is isolated from the solutionby magnetic decantation, which retains all particle sizes, or bycentrifugation and retention of the supernatant, which ensures retentionof only the smallest particle sizes. However, the magnetic material usedin the embodiments described herein preferably does not include suchfine particles.

Substantially eliminating these smallest magnetic particles allowsformation of a microsphere that has relatively high magnetite contentwhile minimizing surface coverage of the core microsphere by themagnetic material. For example, if relatively small magnetic particlesare coupled to the surface of the core microsphere, the particleseffectively form a relatively thin layer across substantially the entirecore microsphere surface resulting in a microsphere with low magneticresponsiveness and little ability for light emission from the core dueto absorption of light by the particles. In one particular example,completely coating the surface of a 7 μm diameter microsphere with 5 nmdiameter magnetite provides a microsphere with only 1% magnetitecontent. In addition, photon transmission into and out of thismicrosphere would be severely inhibited by the magnetite.

In a preferred embodiment, therefore, larger particles, or clusters(i.e., aggregates) of particles, are dispersed across the surface of thecore microsphere such that the entire surface of the core microsphere isnot covered by the magnetic particles. Therefore, the microsphere canhave relatively high magnetic content due to the relatively large sizeof the particles and relatively high light transmission due to thepartial surface coverage of the core microsphere by the magneticparticles. For example, by processing the magnetite to substantiallyexclude smaller particles and to retain particles or clusters havingsizes in a range of about 10 nm to about 1000 nm, and more preferablyabout 50 nm to about 300 nm, it is possible to form a microsphere havingabout 5% magnetite content with only about 20% surface coverage of thecore microsphere by the magnetic material. Microspheres formed accordingto embodiments described herein, therefore, advantageously have highermagnetic content and higher light transmission than magneticmicrospheres formed by methods such as those disclosed by Wang et al.and Sutor, in which the object is to maximize magnetic content withoutregard to the degree of surface coverage or obstruction of light by themagnetic particles.

Treating the core microspheres as described above produces magnetizedcore microspheres, which as shown in FIG. 2, include magnetic material44 coupled to a surface of core microsphere 46. Preferably, about 50% orless of the surface of the core microsphere is covered by magneticmaterial 44. In other words, the magnetic material is coupled to only aportion of the surface of the core microsphere thereby resulting inpartial coverage of the surface by the magnetic material. Such couplingof the magnetic material to the core microsphere is advantageous sincesurface coverage of the core microsphere by the magnetic materialexceeding about 50% has been shown to significantly impact (i.e.,reduce) fluorescent emission from the fluorescently dyed coremicrosphere.

Magnetic material 44 may include particles. In one embodiment, magneticmaterial 44 includes particles having a size (e.g., a diameter) of about10 nm to about 1000 nm. In a preferred embodiment, the magnetic materialincludes particles having a size of about 50 nm to about 300 nm. Thesize of the magnetic particles may be selected based on the size of thecore microsphere, the selected surface coverage of the core microsphereby the magnetic particles, and the selected magnetic content of themicrosphere. In general, the magnetic particles may have a size that issmaller than the size of the core microsphere such that multiplemagnetic particles can be coupled to the surface of the core microspherewithout complete coverage of the surface by the magnetic particles.

In some embodiments, the magnetic material includes single crystals ofmagnetite. In a different embodiment, the magnetic material includesaggregates of particles. The particles that form the aggregates may besmaller than the single crystals of magnetite. In addition, theparticles that form the aggregates may have sizes such that theaggregates themselves have sizes in a range such as those describedabove. In a further embodiment, the magnetic material includes a mixedmetal magnetic material.

The magnetic material may take a number of forms such as ferromagnetic,diamagnetic, paramagnetic, or super-paramagnetic. Of these forms, thelast two are the most useful for embodiments described herein, and sincesuper-paramagnetism is a subclass of paramagnetism, they will be treatedthe same in this discussion. A commonly used magnetic material is theiron oxide magnetite, Fe₃O₄. When such magnetite is prepared by methodsthat produce relatively small particle sizes, the magnetite isparamagnetic. One such method includes heating a solution of iron saltswith a relatively strong base such as sodium hydroxide. One or moreother divalent metals such as cobalt and manganese can be added to theiron salts to form a mixed metal magnetic material with differentproperties. Many of these mixed metal compounds can be used to formmagnetic microspheres. Other magnetic metal oxides are suitable for usein the embodiments described herein. Although some embodiments aredescribed herein with respect to magnetite, any other appropriatemagnetic material may be used in the embodiments described herein.

The method embodiment also includes combining the magnetized coremicrospheres with one or more polymerizable materials such that the oneor more polymerizable materials form a polymer layer surrounding themagnetized core microspheres thereby forming the microspheres thatexhibit magnetic properties. For example, after coupling the magnetiteto the surface of the core microsphere, a protective layer of polymermay be formed over the magnetized core microsphere. In this manner, themagnetically “coated” core microspheres are essentially coated with apolymeric material. The polymer layer may be configured to provide abarrier to prevent (or at least substantially reduce) contact of themagnetic material with the outer environment such as solvents,reactants, analytes, etc. that the formed microspheres may be exposed toin subsequent steps (e.g., dyeing) or during use (e.g., assays). Forexample, as shown in FIG. 3, polymer layer 48 surrounds magneticmaterial 44 and core microsphere 46. Although polymer layer 48 is shownin FIG. 3 as completely surrounding the magnetic material and the coremicrosphere, in practice the polymer layer may not completely surroundthe magnetized core microsphere. However, the polymer layer ispreferably formed such that the polymer layer substantially preventscontact between the magnetic material and other materials that contactthe outer surface of the polymer layer.

This polymer layer can be formed by suspending the magnetized coremicrospheres in a suitable solvent such as water or alcohol and addingto the suspension one or more monomers such as styrene, acrylic acid, orother suitable polymerizable molecules known in the art along with apolymerization initiator and possibly other molecules as appropriate. Itis preferable, although not required, that the monomer mixture includesa species capable of coordinating to the magnetite as well as being ableto copolymerize with the other monomers such as, but not limited to,acrylic acid or vinyl pyridine. This coordination of the polymer layerto the magnetite will form a stronger coupling between the polymer layerand the magnetized core microsphere. The initiator can be a radicalforming compound, a redox pair, or any other appropriate initiator knownin the art. In the absence of an initiator, polymerization can beinduced by any appropriate method known in the art such as ultrasonic orphotochemical initiation. The polymer layer is preferably formed in theabsence of surfactants and polymeric stabilizers, although this is not arequirement of the embodiments described herein.

As described above, one or more functional groups (not shown) may becoupled to the surface of core microsphere 46. In addition, oralternatively, the microspheres may include one or more functionalgroups (not shown) coupled to an outer surface of polymer layer 48. Inparticular, in some embodiments, the method includes coupling one ormore functional groups to an outer surface of the polymer layer. The oneor more functional groups may be coupled to the outer surface of thepolymer layer during polymerization of the one or more polymerizablematerials (e.g., by using one or more polymerizable materials thatinclude one or more functional groups). Alternatively, the one or morefunctional groups may be attached to the outer surface of the polymerlayer after the polymer layer has been formed. Such attachment may beperformed using any suitable method known in the art. The one or morefunctional groups coupled to the outer surface of the polymer layer mayinclude any of the functional groups described above. In addition, ifone or more functional groups are coupled to the surface of the coremicrosphere and the outer surface of the polymer layer, the one or morefunctional groups coupled to the surface of the core microsphere may besubstantially the same as or different than the one or more functionalgroups coupled to the outer surface of the polymer layer.

In one embodiment, the microspheres include one or more fluorochromes(not shown). In another embodiment, the microspheres include two or moredifferent fluorochromes (not shown). Examples of suitablefluorochrome(s) for the microsphere embodiments described herein aredescribed in the patents incorporated by reference herein. Thefluorochrome(s) may be contained in the core microsphere, the polymerlayer, or both the core microsphere and the polymer layer.

In one embodiment, the method includes incorporating one or morefluorochromes into the core microspheres prior to combining the coremicrospheres with the magnetic material. Therefore, the fluorochrome(s)are incorporated into the core microsphere before the polymer layer isformed. In addition, or alternatively, one or more fluorochromes may beattached to the surface of the core microsphere. The fluorochrome(s) maybe attached to the surface of the core microsphere before the polymerlayer is formed.

Additionally, or alternatively, fluorochrome(s) may be incorporated intothe polymer layer and/or attached to the outer surface of the polymerlayer. In this manner, the fluorochromes may be incorporated into themicrosphere after the polymer layer is formed. The fluorochrome(s)incorporated into the polymer layer and/or attached to the outer surfaceof the polymer layer may be the same as or different than thefluorochrome(s) that are incorporated into the core microsphere and/orattached to the surface of the core microsphere. For example, differentfluorochromes may be attached to the surface of the core microsphere andthe outer surface of the polymer layer in different steps. In anotherexample, the same fluorochrome(s) may be incorporated into the coremicrosphere and the polymer layer in the same step. The fluorochrome(s)may be incorporated into and/or attached to the surface of the coremicrosphere and/or the polymer layer using any appropriate method knownin the art.

In one embodiment, the method includes swelling the formed microspheresin a fluorochrome containing solvent such that the fluorochrome migratesinto the formed microspheres. Such a method also includes changing oneor more properties of the fluorochrome containing solvent such that theformed microspheres shrink thereby entrapping the fluorochrome in theformed microspheres. Examples of methods that can be used forincorporating fluorochrome(s) into the microspheres as described aboveare illustrated in U.S. Pat. No. 6,514,295 to Chandler et al., U.S. Pat.No. 6,599,331 to Chandler et al., U.S. Pat. No. 6,632,526 to Chandler etal., and U.S. Pat. No. 6,929,859 to Chandler et al., which areincorporated by reference as if fully set forth herein. The fluorochromecontaining solvent may include one or more fluorochromes or two or moredifferent fluorochromes. The fluorochrome(s) may be entrapped in thecore microspheres and possibly the polymer layer. For example, whetheror not the fluorochrome(s) become entrapped in the polymer layer mayvary depending on the characteristics of the fluorochromes(s) (which maybe selected based on the characteristics of the core microspheres andthe measurement systems and/or methods in which the formed microsphereswill be used), the characteristics of the dyeing solvent, and thecharacteristics of the polymer layer.

In some embodiments, the method includes combining the formedmicrospheres with an additional magnetic material such that theadditional magnetic material couples to an outer surface of the polymerlayer. Such an embodiment may also include forming an additional polymerlayer surrounding the additional magnetic material. In one suchembodiment, additional magnetic material 50 is coupled to an outersurface of polymer layer 48, as shown in FIG. 4. In such an embodiment,as shown in FIG. 5, additional polymer layer 52 may surround additionalmagnetic material 50. Polymer layer 52 may “surround” the additionalmagnetic material as described above. In this manner, the formedmicrospheres can optionally be coated with magnetic material 50 coupledto an outer surface of polymer layer 48, as shown in FIG. 4, followed byanother polymeric coating (e.g., additional polymer layer 52 shown inFIG. 5), to increase the magnetic content of the formed microspheres.This process can be repeated as often as necessary to provide formedmicrospheres with the desired levels of light/fluorescence transmissionand magnetic properties.

Magnetic material 50 may include any of the magnetic materials describedabove. Magnetic material 50 may also be formed as described above. Inaddition, magnetic materials 44 and 50 may have substantially the samecomposition or different compositions. Additional polymer layer 52 maybe formed of any of the polymerizable materials described above.Additional polymer layer 52 may also be formed as described above. Inaddition, polymer layer 48 and additional polymer layer 52 may be formedof substantially the same polymerizable material or differentpolymerizable materials. Furthermore, as shown in FIGS. 4 and 5, theouter surface of polymer layer 48 is only partially covered by magneticmaterial 50. For example, about 50% or less of polymer layer 48 iscovered by the additional magnetic material such that the lighttransmission/fluorescent emission properties of the formed microspheresare not substantially reduced by additional magnetic material 50.

As described above, one or more functional groups (not shown) may becoupled to the surface of core microsphere 46 and/or the outer surfaceof polymer layer 48. In addition, or alternatively, the microspheres mayinclude one or more functional groups (not shown) coupled to an outersurface of additional polymer layer 52. In particular, in someembodiments, the method includes coupling one or more functional groupsto an outer surface of the additional polymer layer. The one or morefunctional groups may be coupled to the outer surface of the additionalpolymer layer during polymerization of the one or more polymerizablematerials (e.g., by using one or more polymerizable materials thatinclude one or more functional groups). Alternatively, the one or morefunctional groups may be attached to the outer surface of the additionalpolymer layer after the additional polymer layer has been formed. Suchattachment of the one or more functional groups may be performed usingany appropriate method known in the art. The one or more functionalgroups coupled to the outer surface of the additional polymer layer mayinclude any of the functional groups described above. In addition, ifone or more functional groups are coupled to the surface of the coremicrosphere and the outer surface of the additional polymer layer, theone or more functional groups coupled to the surface of the coremicrosphere may be substantially the same as or different than the oneor more functional groups coupled to the outer surface of the additionalpolymer layer. If one or more functional groups are coupled to the outersurfaces of the polymer layer and the additional polymer layer, the oneor more functional groups coupled to the outer surface of the polymerlayer may be substantially the same as or different than the one or morefunctional groups coupled to the outer surface of the additional polymerlayer.

As described above, fluorochrome(s) may be incorporated into the coremicrosphere and/or polymer layer 48 and/or may be attached to thesurface of the core microsphere and/or the outer surface of polymerlayer 48. Additionally, or alternatively, fluorochrome(s) may beincorporated into the additional polymer layer and/or attached to theouter surface of the additional polymer layer. The fluorochrome(s)incorporated into the additional polymer layer and/or attached to theouter surface of the additional polymer layer may be the same as ordifferent than the fluorochrome(s) that are incorporated into the coremicrosphere and/or attached to the surface of the core microsphere. Thefluorochrome(s) incorporated into the additional polymer layer and/orattached to the outer surface of the additional polymer layer may alsobe the same as or different than the fluorochrome(s) that areincorporated into the polymer layer and/or attached to the outer surfaceof the polymer layer. The fluorochrome(s) may be incorporated intoand/or attached to the outer surface of the additional polymer layerusing any appropriate method known in the art. Each of the embodimentsof the method described above may include any other step(s) of any othermethod(s) described herein.

FIG. 3 illustrates one embodiment of a microsphere that is configured toexhibit fluorescent and magnetic properties. The microsphere includescore microsphere 46, which may be configured as described above. In oneembodiment, the core microsphere includes one or more functional groups(not shown) coupled to the surface of the core microsphere. The one ormore functional groups may include any of the functional groupsdescribed herein.

The microsphere also includes magnetic material 44 coupled to a surfaceof core microsphere 46. The magnetic material may include any of themagnetic materials described herein and may be configured as describedherein. For example, in one embodiment, the magnetic material includesparticles having a size (e.g., a diameter) of about 10 nm to about 1000nm. In a preferred embodiment, the magnetic material includes particleshaving a size of about 50 nm to about 300 nm. In another embodiment, themagnetic material includes single crystals of magnetite. In a furtherembodiment, the magnetic material includes aggregates of particles. Insome embodiments, the magnetic material includes a mixed metal magneticmaterial. Preferably, about 50% or less of the surface of the coremicrosphere is covered by the magnetic material.

The microsphere further includes polymer layer 48 surrounding themagnetic material and the core microsphere. The polymer layer may beconfigured as described above. In one embodiment, the microsphereincludes one or more functional groups (not shown) coupled to an outersurface of the polymer layer. These one or more functional groups mayinclude any of the functional groups described herein. In addition, iffunctional group(s) are coupled to the surface of the core microsphereand the outer surface of the polymer layer, the functional group(s)coupled to the surface of the core microsphere may be the same as ordifferent than the functional group(s) coupled to the outer surface ofthe polymer layer.

In one embodiment, the microsphere includes an additional magneticmaterial coupled to an outer surface of the polymer layer and anadditional polymer layer surrounding the additional magnetic material.For example, as shown in FIG. 5, the microsphere may include additionalmagnetic material 50 coupled to an outer surface of polymer layer 48 andadditional polymer layer 52 surrounding the additional magneticmaterial. The additional magnetic material may include any of themagnetic materials described herein. In addition, the additionalmagnetic material may be configured as described herein. For example,preferably, the combination of the magnetic material and the additionalmagnetic material covers about 50% or less of the surface of the coremicrosphere. In one embodiment, the magnetic material and the additionalmagnetic material have substantially the same composition. In analternative embodiment, the magnetic material and the additionalmagnetic material have different compositions. In a further embodiment,at least one of the magnetic material and the additional magneticmaterial includes a mixed metal magnetic material.

Additional polymer layer 52 may be formed of any of the polymerizablematerials described herein. In addition, the additional polymer layermay be configured as described herein. Furthermore, in one embodiment,the polymer layer and the additional polymer layer are formed ofsubstantially the same polymerizable material. In an alternativeembodiment, the polymer layer and the additional polymer layer areformed of different polymerizable materials.

In one embodiment, the microsphere includes one or more functionalgroups (not shown) coupled to an outer surface of the additional polymerlayer. These one or more functional groups may include any of thefunctional groups described herein. In addition, if functional group(s)are coupled to the surface of the core microsphere and the outer surfaceof the additional polymer layer, the functional group(s) coupled to thesurface of the core microsphere may be the same as or different than thefunctional group(s) coupled to the outer surface of the additionalpolymer layer. Furthermore, if functional group(s) are coupled to theouter surfaces of the polymer layer and the additional polymer layer,the functional group(s) coupled to the outer surface of the polymerlayer may be the same as or different than the functional group(s)coupled to the outer surface of the additional polymer layer.

In some embodiments, the microsphere includes one or more fluorochromes.In another embodiment, the microsphere includes two or more differentfluorochromes. For example, in one embodiment, one or more fluorochromes(not shown) or two or more different fluorochromes (not shown) may becoupled to a surface of the core microsphere and/or incorporated intothe core microsphere. The fluorochrome(s) coupled to the surface of thecore microsphere and/or incorporated into the core microsphere mayinclude any of the fluorochromes described herein. In anotherembodiment, one or more fluorochromes (not shown) or two or moredifferent fluorochromes (not shown) may be coupled to an outer surfaceof the polymer layer and/or incorporated into the polymer layer. Thefluorochrome(s) coupled to the outer surface of the polymer layer and/orincorporated into the polymer layer may include any of the fluorochromesdescribed herein. In a further embodiment, one or more fluorochromes(not shown) or two or more different fluorochromes (not shown) may becoupled to an outer surface of the additional polymer layer and/orincorporated into the additional polymer layer. The fluorochrome(s)coupled to the outer surface of the additional polymer layer and/orincorporated into the additional polymer layer may include any of thefluorochromes described herein. Furthermore, fluorochrome(s) may beattached to the surface of and/or incorporated into the coremicrosphere, the polymer layer, the additional polymer layer, or somecombination thereof. Each of the embodiments of the microspheredescribed above may be further configured as described herein.

The microsphere embodiments described above provide several advantagesover other currently used microspheres. For example, the microspheredescribed above can include greater than about 2% by weight of themagnetic material without significantly hindering light transmissioninto and out of the microsphere. In particular, it has been determinedthat when more than about 50% of the surface of the core microsphere isobscured by the magnetic material, the fluorescent emission of themicrosphere becomes significantly impacted (i.e., the fluorescentemission is significantly lower). In addition, the magnetic material canbe strongly associated with the surface of the core microsphere asdescribed further herein such that the magnetic content of themicrosphere is not reduced during dyeing operations. The magnetized coremicrosphere is also substantially surrounded by a polymer layer, whichsubstantially prevents the magnetic material from interacting with thebiomolecules of interest and other materials that contact the formedmicrosphere. Furthermore, the polymer layer may be formed in the absenceof surfactants and stabilizers. Therefore, when the microsphereembodiments described herein are used in bioassays, unwantedinterference and changes in the binding efficiency of biomolecules tothe microsphere surface, which may be caused by surfactants andstabilizers, are eliminated. The microsphere embodiments described abovealso have all of the advantages of other embodiments described herein.

The microspheres described herein may be included in a population ofmicrospheres configured to exhibit fluorescent and magnetic properties.For instance, one embodiment of a population of microspheres configuredto exhibit fluorescent and magnetic properties includes two or moresubsets of microspheres. The two or more subsets of microspheres areconfigured to exhibit different fluorescent properties, differentmagnetic properties, or different fluorescent and magnetic properties.The fluorescent and/or magnetic properties of the subsets are preferablysufficiently different across the subsets and sufficiently uniformwithin the subsets such that the fluorescent and/or magnetic properties,when measured, can be used to distinguish the subsets from each other(e.g., to determine the subset to which a microsphere belongs).

Individual microspheres in the two or more subsets may be configured asdescribed herein. For instance, an individual microsphere includes acore microsphere. A magnetic material is coupled to a surface of thecore microsphere. Preferably, about 50% or less of the surface of thecore microsphere is covered by the magnetic material. The individualmicrosphere also includes a polymer layer surrounding the magneticmaterial and the core microsphere. The individual microspheres and thepopulation may be further configured as described herein. Thisembodiment of a population of microspheres has all of the advantages ofother embodiments described herein.

The following examples are not to be considered limiting embodiments ofthe invention and are included herein for example purposes only.

Example 1 Preparation of Magnetite

0.2 moles of Iron (III) chloride hexahydrate and 0.1 mole of iron (II)chloride tetrahydrate were dissolved in 400 ml of deionized water in athree-neck round bottom flask with overhead stirring. This mixture washeated to 90° C. with continued stirring. 520 ml of 6N NaOH was addeddropwise over 1 hour. The reaction was allowed to continue for 24 hours.

Example 2 Magnetite and Polymer Coating

10.2 g of commercially available polystyrene core microspheres with afunctionalized carboxylated surface modification were suspended in atotal volume of 100 ml methanol. 12.4 ml of the prepared magnetitesolution was washed with deionized water, followed by two washes in 1MHCL, and finally two washes with methanol. Each wash step was followedby centrifugation at approximately 4000×g to separate the largermagnetite particles from the smaller magnetic particles. The magnetitewas resuspended in a total volume of 100 ml of methanol. The magnetiteand the core microspheres were combined and allowed to mix for 3 hours.This mixture was then washed four times with deionized water, and thenresuspended in a total volume of 375 ml of water and charged to a 500 mlthree-neck round bottom flask.

A mixture of 12.6 g distilled styrene, 0.768 g distilled divinylbenzene,0.173 g benzoyl peroxide, and 1.47 g acrylic acid was prepared andcharged to the 500 ml three-neck round bottom flask. The reactionmixture was then heated to 60° C. for 24 hours. The coated microsphereswere washed with methanol, tetrahydrofuran, followed by three additionalwashes with methanol, once with water, and finally resuspended indeionized water.

Further modifications and alternative embodiments of various aspects ofthe invention may be apparent to those skilled in the art in view ofthis description. For example, microspheres and populations ofmicrospheres that are configured to exhibit fluorescent and magneticproperties are provided. Accordingly, this description is to beconstrued as illustrative only and is for the purpose of teaching thoseskilled in the art the general manner of carrying out the invention. Itis to be understood that the forms of the invention shown and describedherein are to be taken as the presently preferred embodiments. Elementsand materials may be substituted for those illustrated and describedherein, parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1. A microsphere configured to exhibit fluorescent and magneticproperties, wherein the microsphere is formed in accordance with aprocess comprising: separating magnetic particles by size into a firstgroup and a second group, wherein the magnetic particles in the firstgroup are substantially larger than the magnetic particles in the secondgroup; combining core microspheres with only the magnetic particles inthe first group such that the magnetic particles couple to a surface ofthe core microsphere to form magnetized core microspheres, wherein about50% or less of the surface of the core microspheres is covered by themagnetic particles in the first group; and combining the magnetized coremicrosphere with one or more polymerizable materials such that the oneor more polymerizable materials form a polymer layer surrounding eachmagnetized core microsphere.
 2. The microsphere of claim 1, wherein thecore microspheres comprise one or more functional groups coupled to thesurface of the core microspheres.
 3. The microsphere of claim 1, whereinthe microsphere comprises one or more fluorochromes.
 4. The microsphereof claim 1, wherein the microsphere comprises two or more differentfluorochromes.
 5. The microsphere of claim 1, wherein sizes of themagnetic particles in the first group range between about 10 nm andabout 1000 nm.
 6. The microsphere of claim 1, wherein sizes of themagnetic particles in the first group range between about 50 nm andabout 300 nm.
 7. The microsphere of claim 1, wherein the magneticparticles in the first group comprise single crystals of magnetite. 8.The microsphere of claim 1, wherein the magnetic particles in the firstgroup comprise aggregates of particles.
 9. The microsphere of claim 1,wherein the magnetic particles comprise a mixed metal magnetic material.10. The microsphere of claim 1, wherein the process further comprisescoupling one or more functional groups to an outer surface of thepolymer layer.
 11. The microsphere of claim 1, wherein the processfurther comprises coupling an additional magnetic material to an outersurface of the polymer layer and an additional polymer layer surroundingthe additional magnetic material.
 12. The microsphere of claim 11,wherein the additional magnetic material has substantially the samecomposition as the magnetic particles.
 13. The microsphere of claim 11,wherein the additional magnetic material has a different compositionthan the magnetic particles.
 14. The microsphere of claim 11, whereinthe polymer layer and the additional polymer layer are formed ofsubstantially the same polymerizable material.
 15. The microsphere ofclaim 11, wherein the polymer layer and the additional polymer layer areformed of different polymerizable materials.
 16. The microsphere ofclaim 11, wherein at least one of the magnetic particles and theadditional magnetic material comprises a mixed metal magnetic material.17. The microsphere of claim 11, wherein the process further comprisescoupling or more functional groups to an outer surface of the additionalpolymer layer.
 18. A population of microspheres configured to exhibitfluorescent and magnetic properties, comprising: two or more subsets ofmicrospheres configured to exhibit different fluorescent properties,different magnetic properties, or different fluorescent and magneticproperties, wherein individual microspheres in the two or more subsetsare formed in accordance with a process comprising: separating magneticparticles by size into a first group and a second group, wherein themagnetic particles in the first group are substantially larger than themagnetic second group; combining core microspheres with only themagnetic particles in the first group such that the magnetic particlescouple to a surface of the core microspheres to form magnetized coremicrospheres, wherein about 50% or less of the surface of the coremicrospheres is covered by the magnetic particles in the first group;and combining the magnetized core microspheres with one or morepolymerizable materials such that the one or more polymerizablematerials form a polymer layer surrounding each magnetized coremicrosphere.
 19. A method for forming microspheres that exhibit magneticproperties, comprising: separating magnetic particles by size into afirst group and a second group, wherein the magnetic particles in thefirst group are substantially larger than the magnetic particles in thesecond group; combining core microspheres with only the magneticparticles in the first group such that the magnetic particles couple toa surface of the core microspheres to form magnetized core microspheres,wherein about 50% or less of the surface of the core microspheres iscovered by the magnetic particles in the first group; and combining themagnetized core microspheres with one or more polymerizable materialssuch that the one or more polymerizable materials form a polymer layersurrounding the magnetized core microspheres thereby forming themicrospheres that exhibit magnetic properties.
 20. The method of claim19, wherein a substantial portion of the magnetic particles in the firstgroup have a size of about 10 nm or greater, and wherein a substantialportion of the magnetic particles in the second group have a size ofabout 10 nm or lower.
 21. The method of claim 19, further comprisingcombining the formed microspheres with an additional magnetic materialsuch that the additional magnetic material couples to an outer surfaceof the polymer layer and forming an additional polymer layer surroundingthe additional magnetic material.
 22. The method of claim 19, furthercomprising swelling the formed microspheres in a fluorochrome containingsolvent such that the fluorochrome migrates into the formed microspheresand changing one or more properties of the fluorochrome containingsolvent such that the formed microspheres shrink thereby entrapping thefluorochrome in the formed microspheres.
 23. The method of claim 19,further comprising incorporating one or more fluorochromes into the coremicrospheres prior to combining the core microspheres with the magneticmaterial.
 24. The method of claim 19, further comprising coupling one ormore functional groups to an outer surface of the polymer layer.
 25. Themicrosphere of claim 1, wherein the step of combining the coremicrospheres with only the magnetic particles in the first groupcomprises coupling the magnetic particles in the first group to asurface of the core microspheres via a chemical bond.
 26. Themicrosphere of claim 1, wherein prior to combining the core microsphereswith only the magnetic particles in the first group, the process furthercomprises suspending the magnetic particles in the first group in analcoholic solvent.
 27. The microsphere of claim 1, wherein subsequent tocombining the magnetized core microspheres with one or morepolymerizable materials, the process further comprises swelling theformed microspheres in a fluorochrome containing solvent such that thefluorochrome migrates into the formed microspheres and changing one ormore properties of the fluorochrome containing solvent such that theformed microspheres shrink thereby entrapping the fluorochrome in theformed microspheres.
 28. The microsphere of claim 1, wherein prior tocombining the core microspheres with only the magnetic particles in thefirst group, the process further comprises incorporating one or morefluorochromes into the core microspheres.